Using fluorescence to detect cancerous tissue is not new. R. Alfano et.al.1, 2 first demonstrated that fluorescence can be used to detect cancer in tissue. Ultraviolet (UV) and visible light can be used to excite fluorescence of key molecules to detect cancerous and pre-cancerous tissue from normal tissue or benign tissue. UV luminescence spectroscopy of biomolecules is a powerful tool to study biological specimens, such as bacteria, viruses and biological tissues3-7. UV light in the 250 nm to 400 nm range allows exciting the natural fluorophore in a biological specimen, such as amino acids in proteins and/or NADH. The present invention provides novel methods using phosphorescence data of excited tissue to rapidly detect cancer.
UV illumination in an absorption bands excites molecules of a specimen and radiative relaxation results in light emission, red shifted relative to the absorption wavelength. Emission of light occurs as fluorescence between singlet states and phosphorescence from the triplet to the singlet state. Fast relaxation (˜10−9-10−6 sec) of light emission is associated with fluorescence and longer life-time (˜1 to 10−6 sec) relaxation is associated with phosphorescence.
The delayed phosphorescence is explained by non-radiative relaxation from the singlet excited state S1 into the excited triplet state T1 which is followed by a spin-forbidden transition between T1, and the ground state, S08. Phosphorescence is characterized by the lifetime (τ) and quantum yield (Q) of the transition. The lifetime of phosphorescence is strictly dependent on the fluorophore environment, the rigidity of the structure (motility of the fluorophore), type of solvent and pH. A modified Jablonski diagram is shown in FIG. 1 representing the relationships between singlet and triplet states and the transitions responsible for fluorescence and phosphorescence. The phosphorescence spectrum is red shifted relative to fluorescence spectrum.
The phosphorescence data provide important information on dynamical, geometrical and structural changes in protein structure8, 9. The rigidity of the protein, viscosity of media and low temperatures increase the phosphorescence life-time. The phosphorescence spectra complements the fluorescence data. Using both phosphorescence and fluorescence data of a specimen enhances the detection of changes in biological specimens and/or tissue samples, such as cancerous changes, with a much higher degree of accuracy/sensitivity than fluorescence data alone.
The main components of any biological specimen are proteins. Proteins include the amino-acids bonded together with peptide bonds. The three aromatic amino acids, tryptophan (trp), tyrosine (tyr), and phenylalanine fluoresce in the 300 to 380 nm region, upon excitation in the range from 250 to 300 nm10. Trp, due to its stronger absorption and higher quantum efficiency is the predominate fluorophore in this wavelength range. The main emission band of Trp is in the 300 to 400 nm region centered at about 340 nm. Tyr absorption is blue shifted relative to trp absorption; therefore shorter wavelength excitation will enhance tyr emission. Phenylalanine emission is rarely observed in proteins due to very weak absorption in this range10. The quantum efficiency and Stokes shift of trp fluorescence is highly depend on protein structure and the local environment11, 12. The trp absorption spectrum exhibits structure between 250 and 290 nm with principal maxima at 282 and 288 nm, and a shoulder at 265 nm10. In water, tyr has absorption maxima at 220 and 275 nm13. Tyr fluorescence exhibits a smaller Stokes shift than trp, which is not strongly solvent dependent. The tyr emission maximum is at 305 mm.
Other important natural protein fluorophors in biological tissue are collagen and elastin. The triple helix is a repeating motif in fibril-forming collagens and in range of other extra cellular matrix proteins. This type of structure provided the repeating pattern of (X-Y-Gly)n, where X and Y positions are frequently occupied by Proline (Pro) and Hydroxyproline (Hyp) or Proline (Pro) and Alanine (Ala)14-16. The main absorption band of collagen is located at around 340=n and main fluorescence emission band is at around 380 nm. For elastin the main emission fluorescence band is at 410 nm.
Native fluorescence emission of tissue can be from tryptophan (340 nm), collagen (380 nm), elastin (410 nm), NADH (460 nm) and flavins (525 nm).
Human tissue in general has very specific chemical compositions and the fluorescence and phosphorescence spectral signatures of abnormal tissues will be different from normal tissue. Any changes in the normal composition or structure of the human tissue can affect their fluorescence and phosphorescence emission fingerprints. Here we report on phosphorescence of tissues. According to the present invention, selecting key emission wavelengths from the tissue allows one to make a conclusion regarding the normality of the tissue. In other words, if a tissue changes from normal to pre-cancerous or even cancerous, the fluorescence and phosphorescence emission fingerprints detected using the method of the present invention would indicate the change in the tissue that would allow for the classification of the tissue as cancerous or pre-cancerous.
To avoid experimental imperfections related to measurements of absolute intensity values, the ratio of intensities at the emission key wavelengths are calculated. This ratio is a signature of malignancy or normalcy of the tissue. Based on this ratio, a conclusion on whether a tissue is normal/benign, pre-cancerous or cancerous can be determined.